Summary of the parent Grant. Oxygen radical generation is increased in the postischemic heart and leads to altered nitric oxide (NO) production and postischemic injury. Using Electron Paramagnetic Resonance (EPR) and other techniques, we directly measured the mechanisms of oxygen radical and NO generation in the postischemic heart. From these and other recent studies, it has been shown that oxidants alter NO generation from nitric oxide synthase (NOS). However, the precise role of oxidants in the regulation of NOS structure and function remains unknown. Recently, it has been shown that under ischemic conditions oxygen radical generating enzymes, such as xanthine oxidase (XO), also form NO through reduction of nitrite or nitrate. However, the nature and role of NOS-independent pathways of NO formation in the modulation of postischemic injury is unclear. Therefore, this project will characterize these processes of NO formation and their oxidant interactions. Studies will be performed first at the enzyme level; then in endothelial cells, followed by studies in isolated heart models and then in vivo models of coronary occlusion and reflow. This project has the following 5 specific aims. 1) To characterize mechanisms by which .02- and .02- -derived oxidants affect the structure and function of human endothelial NOS (eNOS). 2) To determine the mechanism by which .02- and .02- -derived oxidants alter eNOS structure and function in the isolated postischemic heart and evaluate approaches to restore eNOS function. 3) To characterize fundamental mechanisms of nitrite, nitrate or organic nitrate mediated NO generation. 4) To characterize the role of nitrite or nitrate mediated pathways of NO generation in the isolated postischemic heart and the mechanisms that regulate this process. 5) In vivo testing and EPR/NMR coimaging of novel therapeutics to inhibit oxidant injury, restore NOS function and confer myocardial protection. For these aims; EPR, electrochemical, and chemiluminescence measurements of oxygen radicals, NO, and NO derived species will be performed along with characterization of the function, structure and modification of the critical NO generating enzymes. Overall, this project will determine the interactions between oxygen radicals and the pathways of NO generation that occur in the process of postischemic injury, and lead to the development of optimal strategies to salvage heart muscle at risk. Supplement Grant Abstract. The central hypothesis of this grant is that changes in endothelial nitric oxide synthase co-factors and substrate availability due to oxidative stress account for endothelial dysfunction and alterations of eNOS function In the post-ischemic heart. Recent studies from our laboratory have shown that tetrahydrobiopterin (BH4) infusion alone or together with the eNOS substrate NADPH have a strong effect on the restoration of coronary flow of hearts subjected to different ischemia times and reperfusion (1). We have also observed that cardiac BH4 and NADPH levels are decreased in the postischemic heart and that eNOS activity decrease parallels the decline in BH4 content (2). In vitro experiments performed with eNOS isolated from control and ischemic hearts showed that BH4 supplementation restores the enzyme activity to control levels and that these effects are dependent on ischemia time (2). The overall aim of this study is to determine the biochemical pathways followed by NADPH and BH4 during ischemia and reperfusion and to evaluate the efficacy of pharmacologic therapies aimed at restoring NOS function and their ability to confer myocardial protection.